Receptor tyrosine kinases from the epidermal growth factor receptor (EGFR) family play critical roles in controlling cellular processes such as proliferation, motility and differentiation. Precise regulation of the spatial and temporal features of EGFR signaling is also very important in tissue patterning during development. Misregulation of EGFR family members is implicated in many human cancers, and two members of this family (EGFR and ErbB2/Her2) are targets of therapeutic agents in current clinical use, including Herceptin, Iressa, and Erbitux. Although much progress has been made in understanding EGFR signaling mechanisms, key questions remain unanswered about how it functions at the cell surface. In particular, pre-formed EGFR dimers, reportedly seen at the cell surface (and important for signaling), have not been described in vitro. Moreover, structural studies of ErbB2 indicate that it is constitutively active, but provide no insight into its regulation at the cell surface. In our studies of invertebrate EGFR orthologs, we have found that C. elegans EGFR forms pre-formed dimers in vitro, offering a unique opportunity to study this key state in detail. Moreover, the D. melanogaster EGFR structurally resembles human ErbB2, yet retains the ability to be regulated directly by growth factor ligands. Scrutinizing the mode of growth factor activation of these invertebrate receptors promises to fill critical gaps in our understanding of the mechanism of EGFR signaling in general. We will also elucidate the molecular mechanisms of two naturally-occurring inhibitors of EGFR signaling (Argos and Kekkon-1) that play critical roles in D. melanogaster tissue patterning. Argos is a secreted protein that neutralizes an activating ligand for Drosophila EGFR. Kekkon-1 is a transmembrane protein that appears to inhibit EGFR directly. By understanding how these inhibitors function, we hope to gain insight for designing novel cancer therapeutics that target EGFR-activating ligands and EGFR respectively. Combining biochemical, biophysical, crystallographic and cellular signaling approaches, we propose to address the following Specific Aims: 1. To determine how growth factor binding regulates the D. melanogaster and C. elegans EGF receptor orthologs, as models for addressing unanswered questions in human ErbB receptor activation. 2. A comparative mechanistic analysis of EGFR signaling inhibition by the secreted inhibitor Argos and the membrane protein Kekkon-1.